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Transcript of Gene Therapy
Current Examples of Gene Therapy
1)Targeting of Acute Myeloid Leukemia Using Chimeric Antigen Receptor Modified T-Cells (June, Grupp)
2) Single Stranded RNAs Used to Allele-Selectively Inhibit Mutant
3) Translating Dosage Compensation to Trisomy 21 (Lawrence, Hall)
An Ethical Discussion
Restoration of "normal" functioning vs. enhancement
How do we decide what is considered normal functioning and what qualifies as a disorder/disability?
Gene Doping (e.g. PPAR-δ, IGF-1, EPO)
Single-stranded RNAs used to Inhibit
Chemotherapy with the goal of reducing the number of leukemic cells from the blood
Induction does not typically kill all leukemia cells and leukemia is likely to return without further treatment
A second round of chemotherapy or a stem cell transplant
High risk of mortality from transplant-related infection or GVH disease
Acute Myeloid Leukemia (AML)
The malignant cell in AML is the
(differentiation arrest and mutations in genes controlling proliferation)
Uncontrolled growth of immature myeloid cells interferes with the production of RBCs, platelets and normal WBCs
Clinical Trial: Emma Whitehead
Diagnosed with leukemia at age 5
Relapsed twice after successive bouts of chemotherapy
First patient to receive genetically modified T-cells to treat leukemia
In remission as of May 2012
Has been used on 12 leukemia patients since Emma
9 of the 12 went into full or partial remission
Using Viral Vectors In Vivo
Can sometimes elicit immune response during delivery
Modify virus capsid to evade immune detection
Helper dependent vectors or "gutless" vector
Some patients remain incurable with current treatment options and may benefit from novel approaches
Ex vivo gene therapy (Cell Therapy)
Targeting CD123 (transmembrane α chain of the interleukin-3 receptor)
Use viral vector to modify patient T-cells
Modified T-cells (CART123) target CD123 of AML or "preleukemic" cells
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MAG. N.p., 25 Jun 2013. Web. 22 Apr 2014. <http://www.hivplusmag.com/case-studies/research-breakthroughs/2013/06/25/it-possible-cure-cancer-hiv-cells>.
Jiang, Jun. "Translating dosage compensation to trisomy 21." Nature.
500.7462 (2013): n. page. Web. 22 Apr. 2014.
June, Carl. "Preclinical targeting of human acute myeloid leukemia and
myeloablation using chimeric antigen receptor–modified T cells." Blood. 123.15 (2014): 2343-2354. Web. 22 Apr. 2014.\
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Fighting Fire with Fire
Single-stranded RNA functions through RNAi in cells
Single-stranded RNA achieves allele-selective inhibition of
Allele-selective inhibition proceeds through a miRNA like mechanism
Direct ssRNA infusion reduces mutant huntingtin levels in mouse brain.
Genetic Correction of Gene Overdose in Trisomy 21
Functional correction of living trisomic cells by inserting a single gene that can epigenetically silence the entire chromosome
Large, noncoding RNA (17 kb) XIST is a natural mechanism used to compensate for the difference in dosage of X-linked genes in females
XIST is produced from the inactive X-chromosome and accumulates across the interphase chromosome structure
XIST RNA induces heterochromatin modification and architectural changes during early development
The X-chromosome is thus transcriptionally silenced and manifests cytologically as a condensed Barr body
Can XIST be inserted into one copy of chromosome 21?
Zinc finger nuclease targeted addition of an inducible XIST transgene
Target is gene-rich core of chromosome 21 in induced pluripotent stem cells derived from patient with Down's syndrome
XIST transgene is considerably large, zinc finger nucleases engineered to a 36 bp sequence of DYRK1A locus at chromosome 21q22
Silencing huntingtin (HTT) expression shows a reversal of phenotype that persists longer than
Allele-selective inhibition maximizes reduction of mutant HTT while minimizing loss of wild-type HTT (could have deleterious effects)
How do we design allele-selective inhibition?
Mutant allele has more CAG repeats (69 mutant/ 17 wt in experimental cell line)
More repeats - formation of hairpin structures, larger structures in mutant allele may be more susceptible to recognition and binding by oligonucleotides
Placement of mismatched bases mimics micro-RNA recognition and optimizes discrimination between mutant and wild-type alleles
Fully complementary duplexes function through a siRNA pathway that involves cleavage of target mRNAs while mismatch-containing duplexes can act through a micro RNA (miRNA)-like pathway that suppresses translation
Duplexes that resemble miRNAs afford greater selectivity
Mismatches introduced into the central region of the dsRNA
The mismatches were at positions predicted to disrupt cleavage of the target by argonaute 2 (AGO2), the mechanism of RNAi
Thus compounds that combine the favorable biodistribution and simpler single-stranded oligonucleotides with the potency of duplex RNAs offers an ideal strategy
Concerns: What about other genes that contain CAG repeats?
No inhibition of TATA-box binding protein (19 CAG repeats), androgen receptor (~20 CAG repeats), etc. at concentrations well above those needed to achieve selective inhibition of mutant HTT.
CART123 orchestrates potent immune response exhibiting antigen-specific degranulation and cytokine production (interferon-γ, macrophage inflammatory protein (MIP), interleukin-2, etc.)
Mouse models engrafted with luciferase-expressing MOLM14 cells, treated with 1 dose of CART123 or control CART19 cells
CD19 is not generally expressed on AML or on normal myeloid cells
Leukemia burden in CART123-treated mice began to diminish within 1 week and was eliminated in most mice within 2 weeks of T-cell infusion
Anti-CD19 CART therapy has been successful in B-cell malignancies due to the following:
Restriction of CD19 to B-cells
Clinical tolerability of prolonged B-cell depletion
Lack of truly tumor-specific antigen in AML
CD123 found on normal AML blasts but also on normal hematopoietec cells
Which virus would make the best vector and why?
Shooting the Messenger
Until now, dsRNA (siRNA) has been used in RNAi-mediated gene silencing
ssRNA too quickly degraded by host enzymes
Does the passenger strand have any unintended side effects?
Poor dispersion of double stranded siRNA requiring various levels of packaging
Combine the dispersion abilities of ss antisense oligonucleotides with the efficiency of RNAi by using ss-silencing RNAs (ss-siRNAs)
ss-siRNAs are chemically modified to provide stability within the host cell
The 5'-thymidine base is modified with an (E)-vinylphosphonate
Mixture of 2’-fluoro (2'-F), 2’-O-methyl (2'-OMe), and 2’-methoxyethyl (2'-MOE) ribose modifications
Trinucleotide repeat disorder which causes the progressive degeneration of nerve cells in the brain
Autosomal dominant pattern of inheritance
The HTT gene is located on the short arm of chromosome 4 at 4p16.3
People with 36 to 39 CAG repeats may or may not develop the signs and symptoms of Huntington disease, while people with 40 or more repeats almost always develop the disorder